The present invention relates generally to the field of biochemistry and medicine and in particular to the cloning, sequencing, and production of contortrostatin and its precursor.
Breast cancer is one of the leading causes of death among non-smoking women and the spread of the disease from the breast to distant sites is a major cause of death in breast cancer patients. At the time of diagnosis over 60% of breast cancer patients will have disease that has spread from the primary site in the breast to some distant site. Spread of cancer to remove sites, e.g. bone, lungs, liver, brain, a process called metastasis, is a characteristic of malignancy and often leads to inoperable disease. Metastasis is the most common factor leading to death from breast cancer. Control of metastasis offers an important avenue for breast cancer treatment. Cancer cells metastasize through the blood or lymph vessels. The first step of metastasis involves the attachment of cancer cells to tissues around the primary site, i.e., to the extracellular matrix (ECM) via cell surface integrins and other adhesion receptors. Integrins mediate cellxe2x80x94cell and cell-substratum interactions and are involved in bi-directional signaling that links the ECM with cytoskeletal proteins. Integrins play an important role in the interaction of mammary carcinoma cells with the ECM. In the second step, cancer cells secrete digestive enzymes that degrade the surrounding tissues allowing the tumor cells to invade these tissues. Eventually, the tumor cells enter the blood or lymphatic system where they repeat the adhesion and invasion steps at a distant (metastatic) site. At this remote site, tumor cells induce the formation of new blood vessels (a process called neovascularization), in and around the growing tumor. These new blood vessels supply nutrients to the metastatic tumor and allow it to grow. Treatments that block any of these steps should act to inhibit metastasis.
Integrins on cancer cells play important roles in tumor invasion and spread. They are a family of proteins found on the cell surface of many cell types that mediate interactions between cells, and between cells and their surroundings. Integrins are heterodimers, composed of xcex1 and xcex2 subunits involved in cellxe2x80x94cell and cell-substratum interactions. Integrins serve as receptors for extracellular matrix proteins such as fibronectin, fibrinogen, vitronectin, collagen and laminen. Some of these interactions have been shown to be mediated via an Arg-Gly-Asp (RGD) sequence present in the matrix proteins. Both the xcex1 and xcex2 subunits are required for fibrinogen binding. For example, one of the members of the superfamily of integrin cell surface receptors is the platelet membrane glycoprotein (GP)IIb/IIIa which interacts with plasma fibrinogen in platelet aggregation.
CN binds to a specific integrin on the surface of blood platelets, and blocks the ability of platelets to adhere to one another (a process called platelet aggregation). Platelets are small fragments of bone marrow cells that are found in the blood stream. They have both beneficial and harmful activities. Their useful action is to stop bleeding following injury by facilitating the formation of a blood clot. But, under certain conditions they are involved in blocking arteries that supply nourishment to the heartxe2x80x94an action that can lead to a heart attack.
Integrin cell surface receptors have been investigated in the role of platelets in mediating coronary artery thrombosis and rethrombosis in the genesis of acute myocardial infarction [Zucker, M. B., Sci. American 242:86 (1990)]. For platelet aggregation an RGD sequence present in fibrinogen is essential for the interaction with (GP)IIb/IIIa [Ginsberg, M. H. et al., Thrombos. Haemostas. 59:1 (1988)]. Because of its inhibition of platelet aggregation, snake venom has been the subject of various investigations.
A number of proteins purified from venom of snakes of the Crotalidae and Viperidae families have been found to inhibit glycoprotein (GP)IIb/IIIa mediated platelet aggregation [see, e.g., Huang, T. F. et al., J. Biol. Chem. 262:16157 (1987); Gan, Z. R. et al., J. Biol. Chem. 263:19827 (1988); Yasuda, T. et al., J. Am. Coll. Cardiol. 16:714 (1990); Trikha, M. et al., Fibrinolysis 4 (Suppl. 1):105 (1990); Trikha, M. et al., Blood 76 (Suppl. 1):479a (1990); Holahan, M. A. et al., Pharmacology 42:340 (1991); Shebuski, R. J. et al., Circulation 82:169 (1990); Yasuda, T. et al., Circulation 83:1038 (1991)]. These proteins, classified as disintegrins, are typically disulfide rich. Moreover, all disintegrins isolated thus far, with the exception of barbourin [Scarborough, R. M. et al., J. Biol. Chem. 266:9359 (1991)] contain an RGD (Arg-Gly-Asp) sequence that has been implicated as being involved in the inhibition of integrin-mediated interactions. In particular, the RGD sequence of the disintegrins may compete for fibrinogen binding sites on the platelet membrane, thereby inhibiting platelet aggregation induced by ADP or other agents.
Nonetheless, there appears to be increasing evidence that disintegrins may have unique surface geometry which facilitates interactions with integrins by mechanisms other than those based solely upon the RGD site. For example, the finding that a mutated, chemically synthesized derivative of echistatin (in which alanine was substituted for arginine in the RGD sequence) still possessed some biological activity, suggests that other regions in the protein may be involved in binding and that there may be some flexibility in the RGD binding site [Connolly, T. M. et al., Circulation 82 (Suppl. III):660 (1990)]. Synthetic RGD peptides, due to their small size, generally do not possess the molecular topography of the disintegrins and therefore cannot interact via the multiplicity of mechanisms likely to be involved in disintegrin binding.
In other investigations, prevention of reocclusion following thrombolysis using tissue-type plasminogen activator in a canine model system has been reported using 30 xcexcg/kg plus 3 xcexcg/kg/min bitistatin, an 83 amino acid disintegrin derived from the venom of Bitis arietans [Shebuski et al., supra], or 15 xcexcg/kg/min i.v. echistatin, a 49 amino acid disintegrin derived from the venom of Echis carinatus [Holahan et al., supra]. In the reported methods, an initial bolus of heparin (100 U/kg i.v.) and subsequent hourly boluses of 50 U/kg were used to increase activated partial thromboplastin times at least 1.5-fold over the control. Whereas it had previously been observed that heparin in combination with tissue-type plasminogen activator (tPA) did not affect the incidence of acute reocclusion in this model system, the addition of echistatin or bitistatin lead to dramatic reductions in the incidence of acute thrombotic reocclusion. The administration of heparin was, however, apparently necessary for prevention of acute thrombotic reocclusion.
Similarly, kistrin (a 68 amino acid disintegrin derived from the venom of Agkistrodon rhodostoma) was evaluated in conjunction with recombinant tissue-type plasminogen activator in a canine model of coronary artery thrombosis with superimposed high grade stenosis [Yasuda et al. (1991), supra]. An effective dose of 4 xcexcg/kg/min was determined to be sufficient to prevent reocclusion. Simultaneous systemic therapeutic heparin anticoagulation was used; the dose of heparin was selected to maintain the activated partial thromboplastin time more than two-fold throughout the experimental observation period.
U.S. Pat. No. 5,066,592 to Huang et al. describes the use of trigramin, a 72 amino acid disintegrin isolated from the venom of Trimeresurus gramineus, to inhibit fibrinogen binding to human platelets and thereby to inhibit fibrinogen-induced aggregation of human platelets. Trigramin is also reported to inhibit binding of von Willebrand factor to platelets. Trigramin is reported to inhibit 125I-fibrinogen binding to ADP (10 xcexcmolar)-stimulated platelets in a concentration-dependent manner with an IC50 of 2.8-5.6xc3x9710xe2x88x928M.
Isolation of an anti-platelet factor applaggin from the venom of Agkistrodon piscivorus piscivorus has also been reported [Chao, B. H. et al., Proc. Natl. Acad. Sci. USA 86:8050 (1989); Savage, B. et al., J. Biol. Chem. 265:11766 (1990)]. Applaggin, unlike trigramin, is reported to inhibit dense-granule secretion in concert with inhibition of platelet aggregation in a dose-dependent manner. While initially described as a homodimer with at least two interchain disulfide bridges [Chao et al. (1989), supra], a subsequent report indicated that analysis of purified applaggin by mass spectroscopy showed the presence of applaggin monomers with a mass of 7,666 Daltons and no evidence of dimerization [Wencel-Drake, J. D. et al., Blood 81:62 (1993)].
One disintegrin of particular interest is CN, which has been isolated from the venom of Agkistrodon contortrix contortrix (the southern copperhead snake). The originally-reported purification procedure included molecular sieve chromatography on Sephadex G-100 SF, desalting on Sephadex G-25F and reverse phase HPLC. ADP-enhanced aggregation of stirred human platelet rich plasma and the inhibition thereof by CN were monitored at 37xc2x0 C. It was found that preincubation for 1 minute of the platelet rich plasma (3xc3x97105/mm3) with 5 xcexcl of the low molecular weight peak after Sephadex G-100 SF resulted in 76% inhibition of platelet aggregation induced by 10 xcexcM ADP [Trikha et al. (1990), supra].
In a subsequent report it was noted that in crude venom, the inhibitor was not readily detectable due to the presence of platelet aggregating activity; however, following the first step of purification (hydrophobic interaction HPLC) inhibitory activity was separated from both aggregating activity and an xcex1-chain degrading fibrinolytic enzyme present in the venom. Inhibitory activity was pooled following HPLC and applied to a hydroxylapatite HPLC column. In the final step of purification, C4 reverse phase HPLC chromatography was employed. The yield of the homogeneous protein was 3-5 mg per gram of venom. CN was reported to have a molecular weight of 18-21 kDa under non-reducing conditions and 9 kDa under reducing conditions; thus, the molecule was believed to be a homodimer with the two subunits being held together by disulfide bond(s). Isoelectric focusing showed that the protein had an acidic pI. CN was reported not to exhibit fibrinolytic activity and was not a 5xe2x80x2-nucleotidase or a phospholipase based on molecular size and kinetics of inhibition of platelet aggregation. Following preincubation for 1 minute, CN at approximately 100 nM was reported to completely inhibit ADP-induced platelet aggregation [Trikha et al. (1990), supra].
It has further been reported that CN has 70 amino acids with five to six disulfide bridges, and that the sequence of CN appears to begin 10 amino acids downstream of applaggin (a platelet aggregation inhibitor from the venom of Agkistrodon piscivorus piscivorus). It was speculated that CN may have an insertion and/or a C-terminal extension of nine amino acids. It was further reported that a 50% inhibition (IC50) of human platelet aggregation in platelet rich plasma was observed at 0.8 xcexcg/ml of CN, and at 2.2 xcexcg/ml with canine platelets [Trikha, M. et al., Journal of Cellular Biochem. 16F:180 (1992)].
CN was reported to inhibit binding of human fibrosarcoma (HT-1080) and c-Ha-ras transfected rat embryo (4R) cells to fibronectin coated plates but not to matrigel coated plates. Inhibition of 4R cell binding to fibronectin in the presence of CN at 1 xcexcg/ml and 5 xcexcg/ml was 46% and 88%, respectively, and for HT1080 cells inhibition was 89% and 85%, respectively [Trikha, M. et al., Proceedings of the American Association for Cancer Research 33:34 (1992)].
Since it appears that CN can inhibit interactions between integrins and their receptors, and may prove useful in the management of diseases associated with these interactions, there exists a need for improved methods to produce greater amounts of purified contortrostatin, substantially free other snake venom components.
The present invention fulfills the need for greater amounts of contortrostatin, which can be used to inhibit biological processes such as platelet aggregation, cell growth, adhesion, metastasis, and neovascularization. Native contortrostatin has been purified and a partial amino acid was determined by Edman degradation. This information enabled a cDNA cloning strategy, which resulted in a full-length cDNA sequence and a deduced amino acid sequence for a contortrostatin pre-cursor protein. The contortrostatin precursor includes a pro-protein region, a metalloproteinase region, and a disintegrin region. The metalloproteinase region includes a metal-binding motif and the disintegrin region includes an RGD loop, which can act as an integrin antagonist. Sequences for native contortrostatin are contained in the distintegrin region.
The present invention includes purified contortrostatin proteins, including the contortrostatin precursor, biologically active variants, and fragments thereof. The contortrostatin protein preferably includes an amino acid sequence that matches native contortrostatin monomer (amino acid numbers 419 to 483 of SEQ ID NO: 2), the metalloproteinase region (amino acid numbers 191 to 410 of SEQ ID NO: 2), the pro-protein region (amino acid numbers 1 to 190 of SEQ ID NO: 2) or the contortrostatin precursor as a whole (SEQ ID NO: 2). A most preferred purified protein is comprised of contortrostatin monomers, each having a molecular mass of about 5 to about 7 kDa, which form a homodimer.
A purified contortrostatin, which can act as an integrin antagonist generally will include in each monomer a constrained Arg-Gly-Asp (RGD) sequence at the tip of a flexible peptide loop of about 13 amino acid residues flanked by two Cys residues, such as the amino acid sequence comprising amino acid numbers 457 to 469 of SEQ ID NO: 2.
Biologically active variants, can include amino acid substitutions, deletions, and insertions, but will generally have an amino acid sequence that is at least 90% homologous to the pro-protein, metalloproteinase, disintegrin and/or contortrostatin regions of the precursor protein. Variants can also include a peptide recognized by an antibody to contortrostatin.
The proteins of the present invention can be made using synthetic methods. The synthetic process can include transcribing and translating a contortrostatin cDNA molecule as disclosed herein, preferably within a transformed host cell. Alternatively, the process involves synthesizing a polypeptide having the amino acid sequence of contortrostatin, as disclosed herein.
Proteins prepared by recombinant DNA methodology will generally include the use of a recombinant DNA molecule comprising a DNA sequence coding on expression for contortrostatin, such as SEQ ID NO:1. Preferably the recombinant DNA molecule encodes sequences having at least one biological activity such as metalloproteinase (e.g. nucleotide numbers 657 to 1316 of SEQ ID NO: 1), or disintegrin (e.g., nucleotide numbers 1341 to 1535 of SEQ ID NO: 1). Moreover, the recombinant DNA molecule can also include pro-protein encoding sequences (e.g., nucleotide numbers 87 to 656 of SEQ ID NO: 1), the entire precursor protein (nucleotide numbers 87 to 1535 of SEQ ID NO: 1), or simply the sequences encoding native contortrostatin monomer (nucleotide numbers 1341 to 1535 of SEQ ID NO: 1).
The present invention further provides a vector, which includes the recombinant DNA molecule, that can be used to transform prokaryotic or eukaryotic host cells. Host cells can be mammalian cells, plant cells, insect cells, yeast and other fungi or bacteria. Processes for producing recombinant contortrostatin will generally include steps, such as culturing the host cell and recovering the contortrostatin expressed by the host cell.
The contortrostatin proteins of the present invention can be formulated as a pharmaceutically acceptable composition, comprising a pharmaceutically acceptable carrier and the purified protein. The pharmaceutical composition can then be used in a method of treating a patient having a disease associated with a ligand binding to an integrin receptor. The treatment generally involves administering the composition to the patient such that integrin binding to integrin receptors is substantially inhibited and the patient is treated. The method of treatment can be used to inhibit platelet aggregation, tumor metastasis, angiogenesis, neovascularization, cell adhesion, invasiveness, or growth.